A 40- to 70-fold increase in erythrocyte adenosine deaminase (ADA) activity has been associated with chronic hemolytic anemia and decreased erythrocyte ATP levels. This disorder is present in 11 of 23 members of an affected kindred, is inherited as an autosomal dominant trait, and is unusual in that the overactivity of ADA is confined to erythrocytes. The kinetic and physiochemical properties of the ADA enzyme in red cells are normal, suggesting the cell-line specific overabundance of a normal enzyme. Since this enzyme is completely stable over the 120 day lifespan of the erythrocyte, this disorder appears to represent a state of true enzyme overproduction rather than of increased enzyme stability. We propose to further define the molecular defect in that kindred by 1) defining the levels of ADA-specific mRNA in erythroid and non-erythroid cells; 2) quantitating the amount of ADA protein in red cells and lymphoblasts from affected individuals; 3) examining DNA from cultured lymphoblasts for altered patterns of restriction endonuclease digestion; 4) constructing a cDNA library from reticulocyte poly A+-selected RNA and cloning the two ADA alleles from this library; 5) sequencing full length ADA clones looking for specific nucleotide insertions, deletions, or substitutions; 6) examining the in vitro translation potential of ADA mRNA obtained from affected reticulocytes or from RNA copies of abnormal cDNA clones; 7) examing the tissue specificity of ADA expression by quantitating the production of ADA enzyme in erythroid and non-erythroid cell lines transfected with ADA cDNAs cloned into a selectable expression vector. These experiments should elucidate a mechanism for the selective overproduction of a specific protein mulecule in a single human cell type. The results of these studies should have important ramifications for the basic understanding of tissue-specific gene expression, as well as for the use of gene transfer techniques in the treatment of congenital enzyme deficiency states.